The broad, long-term goal of this project is to investigate the diffusion and reaction of nitric oxide (NO) in blood, particularly its interaction with red blood cells (RBCs). It is hypothesized that RBCs possess specific mechanisms that regulate the NO consumption rate through modulation of membrane permeability to NO. Specifically the following aims will be pursued. Specific Aim 1: Is NO consumption by RBC regulated by transmembrane diffusion? Specific Aim 2: Do any specific intra- erythrocytic molecules participate in the regulation of NO quenching? Specific Aim 3: How does the regulation of NO consumption by RBCs affect vessel regulation? The first two aims will be addressed by use of a competitive experiment and a differential membrane bioreactor specifically designed to measure the NO-RBC reaction rate. Kinetic models will be used to analyze the data. Biophysical (EPR and fluorescence) and biochemical (characterization of enzymes, metabolites, and lipids) techniques will be applied to RBCs, RBC ghosts, and synthetic liposomes in order to answer these questions. The last aim will be addressed using isolated porcine coronary microvessels as a bio-assay to determine the functional role of NO quenching and its regulation. The hypotheses proposed above are a significant departure from the current understanding that NO consumption is not regulated and that the RBC membrane is "completely permeable" to NO. In addition to its contribution to fundamental physiology, the proposed work directly impacts multiple aspects of clinical medicine, including NO inhalation therapy and the design of blood substitutes. Furthermore, the proposed mechanism might contribute to the pathology of several diseases, such as essential and pulmonary hypertension, peripheral vascular disease associated with diabetes mellitus, sickle cell anemia, and other hereditary RBC disorders. In these situations, altered RBC membrane consumption by RBCs is essential to the development of clinical intervention and understanding of the complex roles that NO plays under physiological and pathological conditions.